G-protein coupled receptors (GPCRs) are proteins that interact with G-proteins to transmit an intracellular signal. Upon ligand binding, GPCRs trigger the hydrolysis of GTP to GDP by G-protein subunits; GTP hydrolysis is accompanied by a switch from activity to inactivity. It is estimated that there are roughly 1,000 GPCRs [Clapham, Nature 379:297-299 (1996)] and all characterized to date include a seven transmembrane domain that anchors the receptor to the cell. GPCRs include receptors for opiates, adrenaline, histamine, polypeptide hormones, and photons, among other ligands. These receptors are coupled to a wide variety of cellular second messenger pathways including, for example, pathways that alter intracellular calcium concentrations and cAMP levels.
Among the various GPCRs identified, CD97 appears to be representative of a sub-family of proteins which effect cellular adhesion [McKnight, et al., Immunol Today 17:283-287(1996)]. CD97 and related receptors are unique in that their structure includes a transmembrane domain that directly links a cytoplasmic domain that participates in GTP hydrolysis with extracellular protein binding domains that specifically participate in cell-cell adhesion. The extracellular, amino terminal region of CD97 includes numerous cell-cell adhesive motifs, including multiple epidermal growth factor-like (EGF-like) repeats and an integrin binding site [Hamann J, et al., Immunol 155:1942xe2x80x941950 (1996); Gray, et al., J. Immunol 157:5438-5447 (1996)]. Proteins that contain EGF-like repeats have been shown to be involved in cell adhesion events [Campbell, et al., Curr. Opin. Struct. Biol. 3:385-392 (1993); Rao,etal., Cell 82:131-141 (1995)], and consistent with this observation, heterologous expression of CD97 in COS cells elicits homotypic cell aggregation that can be blocked in the presence of anti-CD97 monoclonal antibodies [Hamann, et al., J. Exp. Med. 184:1185-1189 (1996)]. CD97 and related proteins have been referred to as the EGF-7TM subfamily of seven transmembrane receptors [McKNight and Gordon, Immunol. Today 17:283-2887 (1996)]. Ligands identified for CD97 include members of the integrin family of cell surface adhesion receptors. Various integlins recognize and interact with their cognate ligands through a trimeric amino acid sequence of arginine-glycine-aspartic acid (denoted RGD in the single letter amino acid code) [D""Souza, etal., Trends Biochem. Sci., 16:246-250 (1991)] and this sequence has been identified in the extracellular region of CD97, between the EGF-like repeats and the transmembrane domain.
CD97 has been shown to undergo post-translational proteolytic processing which results in an extracellular (and potentially soluble) alpha subunit and a smaller, integral membrane beta subunit [Gray, et al., J. Immunol. 157:5438-5447 (1996)]. The two subunits are associated in a non-covalent manner and the alpha subunit is held at the cell surface through its interaction with the beta subunit. The role of proteolysis is unclear, but it may be a mechanism for receptor down-regulation which is common among proteins, such as selectins and intercellular adhesion molecules (ICAMs), that participate in cell adhesion.
Other members of the CD97 sub-family of GPCRs have been identified by amino acid sequence and structural homology and include human EMR1, HE6, BAI1, the calcium-independent receptor of latrotoxin (CIRL), latrophilin, and proteins encoded by the Caenorhabditis elegans open reading frames designated B0457.1 and B0286.2 [Baud V, et al., Genomics 26:334-344 (1995); McKnight, et al., J. Biol. Chem. 271:486-489 (1996); Krasnoperov, et al., Neuron 18:925-937 (1997); Lelianova, et al., J. Biol. Chem. 272:21504-21508 (1997); Davletov, et al., J. Biol. Chem. 271:23239-23245 (1996); Nishimori, et al., Oncogene 15:2145-2150 (1997)]. EMR1, and its murine homolog F4/80, are macrophage-specific in expression and structurally related to CD97 in that they contain multiple extracellular EGF-like repeats, a rod-like stalk region, and the characteristic transmembrane domain of GPCRs [Baud V, et al., Genomics 26:334-344 (1995); McKnight, et al., J. Biol. Chem. 271:486-489 (1996)]. No ligands have been identified for EMR-1 and it is uncertain if the protein undergoes post-translational proteolytic processing.
CIRL [Krasnoperov, et al., Neuron 18:925-937 (1997); Lelianova, et al., J. Biol. Chem. 272:21504-21508 (1997); Davletov, etal., J. Biol. Chem. 271:23239-23245 (1996)] is believed to be expressed specifically in the central nervous system at neuronal presynaptic terminals and, like CD97, undergoes proteolytic cleavage resulting in an extracellular alpha subunit in non-covalent association with an integral membrane seven-transmembrane beta subunit. Cleavage of latrophilin is believed to occur at a Ser-His-Leu/Thr-Asn-Phe site that is conserved in CD97 [Krasnoperov, et al., Neuron 18:925-937 (1997)]. CIRL has been shown to bind latrotoxin, a component of black widow spider venom, in the 0.5 to 1.0 nM range, and binding of the ligand to CIRL expressed in bovine chromaffin cells has been shown to result in exocytosis, a hallmark of toxin binding [Krasnoperov, et al., Neuron 18:925-937 (1997)]. Alpha latrotoxin binding has also been demonstrated at neuromuscular motor endplates, and this interaction elicits explosive secretory granule release of acetylcholine from presynaptic granules, resulting in muscle paralysis characteristic of the spider""s bite [Petrenko, et al., F.E.B.S. Letts. 325:81-85 (1993)]. It is unclear, however, if the peripheral toxin effects result from binding to CIRL or some other related protein.
Thus there exists a need in the art to identify and characterize other members of the CD97-like family of GPCRs, in particular human receptors which participate in cellular adhesion and those that participate in cytoplasmic metabolic pathways modulated by extracellular signals. Identification of CD97-like receptors can permit identification and diagnosis of disease states which arise from aberrant signaling by the receptor, as well as disease states that arise from aberrant expression of the receptor itself.
The present invention provides purified and isolated human seven transmembrane receptor lectomedin polypeptides or fragments thereof, said polypeptides comprising extracellular lectin-binding, olfactomedin-like, and mucin-like domains. Mature lectomedin polypeptides are also provided wherein signal or leader sequences are cleaved. Preferred polypeptides of the invention comprise the amino acid sequence set out in SEQ ID NO: 2 or a fragment thereof, the amino acid sequence set out in SEQ ID NO: 4 or fragment thereof the amino acid sequence set out in SEQ ID NO: 6 or fragment thereof, and the amino acid sequence set out in SEQ ID NO: 58 or fragment thereof.
The invention also provides polynucleotides encoding polypeptides of the invention. Preferred polynucleotides comprising the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 57. The invention also provides polynucleotides a) encoding a human lectomedin polypeptide selected from the group consisting of the polynucleotide set out in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 57; b) a DNA which hybridizes under moderately stringent conditions to the non-coding strand of the polynucleotide of (a); and c) a DNA which would hybridize to the non-coding strand of the polynucleotide of (a) but for the redundancy of the genetic code. Preferred polynucleotides of the invention are DNA molecules. Preferred DNA molecules are cDNA molecules and genomic DNA molecules. The invention also provides DNA which is a wholly or partially chemically synthesized. Anti-sense polynucleotide which specifically hybridizes with a polynucleotide of the invention are also comprehended.
The invention also proved expression construct comprising the a polynucleotide of the invention, as well as host cells transformed or transfected with a polynucleotide or expression construct of the invention.
The invention also provides polynucleotide of the invention operably linked to a heterologous promoter, and host cells polynucleotides operably linked to a heterologous promoter.
In another aspect, the invention provides methods for producing a human lectomedin polypeptide comprising the steps of: a) growing the host cell of the invention under conditions appropriate for expression of the lectomedin polypeptide and b) isolating the lectomedin polypeptide from the host cell or the medium of its growth.
The invention also proved antibodies specifically immunoreactive with a polypeptide of the invention. Preferably, antibodies of the invention are monoclonal antibodies. The invention also provides cells, e.g. hybridomas, that produce antibodies of the invention. Anti-idiotype antibodies specifically immunoreactive with an antibody of the invention are also comprehended.
The invention also provides methods to identify a specific binding partner compound of a lectomedin polypeptide comprising the steps of: a) contacting the lectomedin polypeptide with a compound under conditions which permit binding between the compound and the lectomedin polypeptide; b) detecting binding of the compound to the lectomedin polypeptide; and c) identifying the compound as a specific binding partner of the lectomedin polypeptide. Methods of the invention embrace specific binding partner that modulate activity of the lectomedin polypeptide. In one aspect, the compound inhibits activity of the lectomedin polypeptide, and in another aspect, the compound enhances activity of the lectomedin polypeptide.
The invention also provides methods to identify a specific binding partner compound of a lectomedin polynucleotide comprising the steps of: a) contacting the lectomedin polynucleotide with a compound under conditions which permit binding between the compound and the lectomedin polynucleotide; b) detecting binding of the compound to the lectomedin polynucleotide; and c) identifying the compound as a specific binding partner of the lectomedin polynucleotide. Methods of the invention embrace specific binding partner that modulates expression of a lectomedin polypeptide encoded by the lectomedin polynucleotide. In one aspect, the compound inhibits expression of the lectomedin polypeptide, and in another aspect, the compound enhances expression of the lectomedin polypeptide. The invention also provides compounds identified by a method of the invention.
In another aspect, the invention comprehends composition comprising the compound identified by a method of the invention and a pharmaceutically acceptable carrier. The invention also provides use of a compound identified by a method of the invention for the preparation of a medicament to treat lectomedin related pathologies.
The invention also provides for use of a lectomedin polypeptide in the preparation of a medicament for the treatment of a lectomedin related disorder.
The present invention provides purified and isolated polypeptides and underlying polynucleotides for a novel family of transmembrane proteins designated lectomedins. The invention includes both naturally occurring and non-naturally occurring lectomedin polynucleotides and polypeptide products thereof. Naturally occurring lectomedin products include distinct gene and polypeptide species within the lectomedin family, including, for example, allelic variants, which are expressed within cells of the same animal, as well as corresponding species homologs expressed in cells of other animals. The invention further provides splice variants encoded by the same polynucleotide but which arise from distinct mRNA transcripts. Non-naturally occurring lectomedin products include variants of the naturally occurring products such as analogs, fragments, fusion (or chimeric) proteins, and lectomedin products having covalent modifications. The lectomedin family of proteins is distinguished from previously known seven transmembrane families of proteins in that the lectomedin proteins include at least one extracellular lectin binding-like domain and at least one extracellular olfactomedin domain. Unlike many other seven transmembrane proteins, the structure of proteins in the lectomedin family of proteins does not include EGF-like binding domains which effect cell/cell interactions. In a preferred embodiment, the invention provides polynucleotides comprising the sequences set forth in SEQ ID NOs: 1, 3, 5 and 57. The invention also embraces polynucleotides encoding the amino acid sequences set out in SEQ ID NOs: 2, 4, 6, and 58. Presently preferred polypeptides of the invention comprises the amino acid sequences set out in SEQ ID NOs: 2, 4, 6, and 58.
The invention also provides expression constructs (or vectors) comprising polynucleotides of the invention, as well as host cells transformed, transfected, or electroporated to include a polynucleotide or expression construct of the invention. Methods to produce a polypeptide of the invention are also comprehended. The invention further provides antibodies, preferably monoclonal antibodies, specifically immunoreactive with a polypeptide of the invention, as well as cell lines, e.g., hybridomas, that secrete the antibodies.
The present invention provides novel purified and isolated human polynucleotides (e.g., DNA sequences and RNA transcripts, both sense and complementary antisense strands, including splice variants thereof) encoding the human lectomedins. DNA sequences of the invention include genomic and cDNA sequences as well as wholly or partially chemically synthesized DNA sequences. Genomic DNA of the invention comprises the protein coding region for a polypeptide of the invention and includes allelic variants of the preferred polynucleotides of the invention. Genomic DNA of the invention is distinguishable from genomic DNAs encoding polypeptides other than lectomedin in that it includes the lectomedin coding region found in lectomedin cDNA of the invention. Genomic DNA of the invention can be transcribed into RNA, and the resulting RNA transcript may undergo one or more splicing events wherein one or more introns (i.e., non-coding regions) of the transcript are removed, or xe2x80x9cspliced out.xe2x80x9d RNA transcripts that can be spliced by alternative mechanisms, and therefore be subjected to removal of different non-coding RNA sequences but still encode a lectomedin polypeptide, are referred to in the art as splice variants, which are embraced by the invention. Splice variants comprehended by the invention, therefore, are encoded by the same DNA sequences but arise from distinct mRNA transcripts. Allelic variants are known in the art to be modified forms of a wild type (predominant) gene sequence, the modification resulting from recombination during chromosomal segregation or exposure to conditions which give rise to genetic mutation. Allelic variants, like wild type genes, are inherently naturally occurring sequences (as opposed to non-naturally occurring variants which arise from in vitro manipulation).
The invention also comprehends cDNA that is obtained through reverse transcription of an RNA polynucleotide encoding lectomedin, followed by second strand synthesis of a complementary strand to provide a double stranded DNA. xe2x80x9cChemically synthesizedxe2x80x9d as used herein and understood in the art, refers to polynucleotides produced by purely chemical, as opposed to enzymatic, methods. xe2x80x9cWhollyxe2x80x9d synthesized DNA sequences are therefore produced entirely by chemical means, and xe2x80x9cpartiallyxe2x80x9d synthesized DNAs embrace those wherein only portions of the resulting DNA were produced by chemical means.
Preferred DNA sequences encoding human lectomedin polypeptides are set out in SEQ ID NOs: 1, 3, 5, and 57. The worker of skill in the art will readily appreciate that preferred DNAs of the invention comprise double stranded molecules, for example, the molecule having the sequence set forth in either SEQ ID NOs: 1, 3, 5, or 57, along with the complementary molecule (the xe2x80x9cnon-coding strandxe2x80x9d or xe2x80x9ccomplementxe2x80x9d) having a sequence deducible from the sequence of SEQ ID NO: 1 according to Watson-Crick base pairing rules for DNA. Also preferred are polynucleotides encoding the lectomedin polypeptides of SEQ ID NOs: 2, 4, 6, and 58.
The invention further embraces species, preferably mammalian, homologs of the human lectomedin DNA. Species homologs, in general, share at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% homology with human DNA of the invention. Percent sequence xe2x80x9chomologyxe2x80x9d with respect to polynucleotides of the invention is defined herein as the percentage of nucleotide bases in the candidate sequence that are identical to nucleotides in the lectomedin coding sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.
The polynucleotide sequence information provided by the invention makes possible large scale expression of the encoded polypeptide by techniques well known and routinely practiced in the art. Polynucleotides also permit identification and isolation of polynucleotides encoding related lectomedin polypeptides by well known techniques including Southern and/or Northern hybridization, and polymerase chain reaction (PCR), ligase chain reaction, as well as other amplification techniques. Examples of related polynucleotides include human and non-human genomic sequences, including allelic variants, as well as polynucleotides encoding polypeptides homologous to lectomedins and structurally related polypeptides sharing one or more biological, immunological, and/or physical properties of lectomedin.
The disclosure of full length polynucleotides encoding lectomedin polypeptides makes readily available to the worker of ordinary skill in the art every possible fragment of the full length polynucleotides. The invention therefore provides fragments of lectomedin coding polynucleotides. Such fragments comprise at least 10 to 20, and preferably at least 15, consecutive nucleotides of the polynucleotide. The invention comprehends, however, fragments of various lengths. Preferably, fragment polynucleotides of the invention comprise sequences unique to the lectomedin coding polynucleotide sequence, and therefore hybridize under highly stringent or moderately stringent conditions only (i.e., xe2x80x9cspecificallyxe2x80x9d) to polynucleotides encoding lectomedin, or lectomedin fragments thereof containing the unique sequence. Polynucleotide fragments of genomic sequences of the invention comprise not only sequences unique to the coding region, but also include fragments of the full length sequence derived from introns, regulatory regions, and/or other non-translated sequences. Sequences unique to polynucleotides of the invention are recognizable through sequence comparison to other known polynucleotides, and can be identified through use of alignment programs routinely utilized in the art, e.g., those made available in public sequence databases.
The invention also provides fragment polynucleotides that are conserved in one or more polynucleotides encoding members of the lectomedin family of polypeptides. Such fragments include sequences characteristic of the family of lectomedin polynucleotides, and are also referred to as xe2x80x9csignature sequences.xe2x80x9d The conserved signature sequences are readily discernable following simple sequence comparison of polynucleotides encoding members of the lectomedin family. Fragments of the invention can be labeled in a manner that permits their detection, including radioactive and non-radioactive labeling.
Fragment polynucleotides are particularly useful as probes for detection of full length or other fragment lectomedin coding polynucleotides. One or more fragment polynucleotides can be included in kits that are used to detect the presence of a polynucleotide encoding lectomedin, or used to detect variations in a polynucleotide sequence encoding lectomedin.
The invention also embraces DNA sequences encoding lectomedin species which hybridize under moderately or highly stringent conditions to the non-coding strand, or complement, of the polynucleotide in SEQ ID NOs: 1, 3, 5, or 57. DNA sequences encoding lectomedin polypeptides which would hybridize thereto but for the redundancy of the genetic code are further comprehended by the invention. Exemplary highly stringent conditions are include hybridization at 45xc2x0 C. in 5xc3x97SSPE and 45% formamide, and a final wash at 65xc2x0 C. in 0.1xc3x97SSC. Exemplary moderately stringent condition include a final wash at 55xc2x0 C. in 1xc3x97SSC. It is understood in the art that conditions of equivalent stringency can be achieved through variation of temperature and buffer, or salt concentration as described Ausubel, et al. (Eds.), Protocols in Molecular Biology, John Wiley and Sons (1994), pp.6.0.3 to 6.4.10. Modifications in hybridization conditions can be empirically determined or precisely calculated based on the length and the percentage of guanosine/cytosine (GC) base pairing of the probe. The hybridization conditions can be calculated as described in Sambrook, et al., (Eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.5 1.
Autonomously replicating recombinant expression constructs such as plasmid and viral DNA vectors incorporating lectomedin coding sequences are also provided. Expression constructs wherein lectomedin-encoding polynucleotides are operably linked to an endogenous or exogenous expression control DNA sequence and a transcription terminator are also provided. Expression control DNA sequences include promoters, enhancers, and operator, and are generally selected based on the expression systems in which the expression construct is to be utilized. Preferred promoter and enhancer sequences are generally selected for the ability to increase gene expression, while operator sequences are generally selected for the ability to regulate gene expression. Expression constructs of the invention may also include sequences encoding one or more selectable markers that permit identification of host cells bearing the construct. Expression constructs may also include sequences that facilitate, and preferably promote, homologous recombination in a host cell. Preferred constructs of the invention also include sequences necessary for replication in a host cell. Expression constructs are preferably utilized for production of an encoded lectomedin protein, but may also be utilized to amplify the construct itself.
According to another aspect of the invention, host cells are provided, including prokaryotic and eukaryotic cells, comprising a polynucleotide of the invention in a manner which permits expression of the encoded lectomedin polypeptide. Polynucleotides of the invention may be introduced into the host cell as part of a circular plasmid, or as linear DNA comprising an isolated protein coding region or a viral vector. Methods for introducing DNA into the host cell well known and routinely practiced in the art include transformation, transfection, electroporation, nuclear injection, or fusion with carriers such as liposomes, micelles, ghost cells, and protoplasts. Expression systems of the invention include bacterial, yeast, fungal, plant, insect, invertebrate, and mammalian cells systems. Host cells of the invention are a valuable source of immunogen for development of antibodies specifically immunoreactive with lectomedin. Host cells of the invention are also useful in methods for large scale production of lectomedin polypeptides wherein the cells are grown in a suitable culture medium and the desired polypeptide products are isolated from the cells or from the medium in which the cells are grown by purification methods known in the art, e.g., conventional chromatographic methods including immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion filtration, cation or anion exchange chromatography, high pressure liquid chromatography (HPLC), reverse phase HPLC and the like. Still other methods of purification include those wherein the desired protein is expressed and purified as a fusion protein having a specific tag, label, or chelating moiety that is recognized by a specific binding partner or agent. The purified protein can be cleaved to yield the desired protein, or be left as an intact fusion protein. Cleavage of the fusion component may produce a form of the desired protein having additional amino acid residues as a result of the cleavage process.
Knowledge of lectomedin coding DNA sequences allows for modification of cells to permit, or increase, expression of endogenous lectomedin. Cells can be modified (e.g., by homologous recombination) to provide increased lectomedin expression by replacing, in whole or in part, the naturally occurring lectomedin promoter with all or part of a heterologous promoter so that the cells express lectomedin at higher levels. The heterologous promoter is inserted in such a manner that it is operably linked to lectomedin-encoding sequences. See, for example, PCT International Publication No. WO 94/12650, PCT International Publication No. WO 92/20808, and PCT International Publication No. WO 91/09955. It is also contemplated that, in addition to heterologous promoter DNA, amplifiable marker DNA (e.g., ada, dhfr, and the multifunctional CAD gene which encodes carbamyl phosphate synthase, aspartate transcarbamylase, and dihydroorotase) and/or intron DNA may be inserted along with the heterologous promoter DNA. If linked to the lectomedin coding sequence, amplification of the marker DNA by standard selection methods results in co-amplification of the lectomedin coding sequences in the cells.
The DNA sequence information provided by the present invention also makes possible the development through, e.g. homologous recombination or xe2x80x9cknock-outxe2x80x9d strategies [Capecchi, Science 244:1288-1292 (1989)], of animals that fail to express functional lectomedin or that express a variant of lectomedin. Such animals are useful as models for studying the in vivo activities of lectomedin and modulators of lectomedin.
The invention also provides purified and isolated mammalian lectomedin polypeptides encoded by a polynucleotide of the invention. Presently preferred are lectomedin polypeptides comprising the amino acid sequence set out in SEQ ID NO: 2, 4, 6, or 58. The invention also embraces lectomedin polypeptides encoded by a DNA selected from the group consisting of: a) the DNA sequence set out in SEQ ID NO: 1, 3, 5 or 57; b) a DNA molecule which hybridizes under high stringent conditions to the noncoding strand of the protein coding portion of (a); and c) a DNA molecule that would hybridize to the DNA of (a) but for the degeneracy of the genetic code.
The invention also embraces variant (or analog) lectomedin polypeptides. In one example, insertion variants are provided wherein one or more amino acid residues supplement a lectomedin amino acid sequence. Insertions may be located at either or both termini of the protein, or may be positioned within internal regions of the lectomedin amino acid sequence. Insertional variants with additional residues at either or both termini can include for example, fusion proteins and proteins including amino acid tags or labels.
In another aspect, the invention provides deletion variants wherein one or more amino acid residues in a lectomedin polypeptide are removed. Deletions can be effected at one or both termini of the lectomedin polypeptide, or with removal of one or more residues within the lectomedin amino acid sequence. Deletion variants, therefore, include all fragments of a lectomedin polypeptide.
In still another aspect, the invention provides substitution variants of lectomedin polypeptides. Substitution variants include those polypeptides wherein one or more amino acid residues of a lectomedin polypeptide are removed and replaced with alternative residues. In one aspect, the substitutions are conservative in nature, however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in Tables A, B, or C below.
The invention also provides derivatives of lectomedin polypeptides. Derivatives include lectomedin polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. Preferably, the modifications are covalent in nature, and include, for example, chemical bonding with polymers, lipids, non-naturally occurring amino acids, other organic, and inorganic moieties. Derivatives of the invention may be prepared to increase circulating half-life of a lectomedin polypeptide, or may be designed to improve targeting capacity for the polypeptide to desired cells, tissues, or organs.
The invention also embraces polypeptides have at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55% or at least 50% identity and/or homology to the preferred polypeptide of the invention. Percent amino acid sequence xe2x80x9cidentityxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the lectomedin sequence after aligning both sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent sequence xe2x80x9chomologyxe2x80x9d with respect to the preferred polypeptide of the invention is defined herein as the percentage of amino acid residues in the candidate sequence that are identical with the residues in the lectomedin sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and also considering any conservative substitutions as part of the sequence identity.
In one aspect, percent homology is calculated as the percentage of amino acid residues in the smaller of two sequences which align with identical amino acid residue in the sequence being compared, when four gaps in a length of 100 amino acids may be introduced to maximize alignment [Dayhoff, in Altas of Protein Sequence and Structure, Vol. 5, p. 124, National Biochemical Research Foundation, Washington, D.C. (1972), incorporated herein by reference].
Polypeptides of the invention may be isolated from natural cell sources or may be chemically synthesized, but are preferably produced by recombinant procedures involving host cells of the invention. Use of mammalian host cells is expected to provide for such post-translational modifications (e.g., glycosylation, truncation, lipidation, and phosphorylation) as may be needed to confer optimal biological activity on recombinant expression products of the invention. Glycosylated and non-glycosylated form of lectomedin polypeptides are embraced.
Insertion variants include lectomedin polypeptides wherein one or more amino acid residues are added to a lectomedin acid sequence, or fragment thereof. Variant products of the invention also include mature lectomedin products, i.e., lectomedin products wherein leader or signal sequences are removed, with additional amino terminal residues. The additional amino terminal residues may be derived from another protein, or may include one or more residues that are not identifiable as being derived from a specific proteins. Lectomedin products with an additional methionine residue at position xe2x88x921 (Metxe2x88x921-lectomedin) are contemplated, as are lectomedin products with additional methionine and lysine residues at positions xe2x88x922 and xe2x88x921 (Metxe2x88x922-Lysxe2x88x921-lectomedin). Variants of lectomedin with multiple, additional Met, Met-Lys, Lys residues are particularly useful for enhanced recombinant protein production in bacterial host cell.
The invention also embraces lectomedin variants having additional amino acid residues which result from use of specific expression systems. For example, use of commercially available vectors that express a desired polypeptide as part of glutathione-S-transferase (GST) fusion product provides the desired polypeptide having an additional glycine residue at position xe2x88x921 after cleavage of the GST component from the desired polypeptide. Variants which result from expression in other vector systems are also contemplated.
Insertional variants also include fusion proteins wherein the amino and/or carboxy termini of the lectomedin polypeptide is fused to another polypeptide. Examples of such fusion proteins are immunogenic polypeptides, proteins with long circulating half life, such as immunoglobulin constant regions, marker proteins (e.g., fluorescent) and proteins or polypeptide that facilitate purification of the desired lectomedin polypeptide, e.g. FLAG(copyright) tags or polyhistidine sequences.
Deletion variants include lectomedin polypeptides wherein one or more amino acid residues are deleted from the lectomedin amino acid sequence. Deletion variants of the invention embrace polypeptide fragments of the sequence set out in SEQ ID NO: 2, 4, 6, or 58 wherein the fragments maintain biological or immunological properties of a lectomedin polypeptide. Fragments comprising at least 5, 10, 15, 20, 25, 30, 35, or 40 consecutive amino acids of SEQ ID NO: 2, 4, 6, or 58 are comprehended by the invention. Preferred polypeptide fragments display antigenic properties unique to or specific for the lectomedin family of polypeptides. Fragments of the invention having the desired biological and immunological properties can be prepared by any of the methods well known and routinely practiced in the art.
Substitution variants of the invention include lectomedin polypeptides, or fragments thereof wherein one or more amino acid residues in the lectomedin amino acid sequence are deleted and replaced with another amino acid residue. Variant polypeptides include those wherein conservative substitutions have been introduced by modification of polynucleotides encoding polypeptides of the invention. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A (from WO 97/09433, page 10, published Mar. 13, 1997 (PCT/GB96/02197, filed Sep. 6, 1996), immediately below.
Alternatively, conservative amino acids can be grouped as described in Lehninger, [Biochemistry, Second Edition; Worth Publishers, Inc. NY:N.Y. (1975), pp.71-77] as set out in Table B, immediately below.
As still an another alternative, exemplary conservative substitutions are set out in Table C, below.
The invention further embraces lectomedin products, or fragments thereof, covalently modified or derivatized, e.g., to include one or more water soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. Particularly preferred are lectomedin products covalently modified with polyethylene glycol (PEG) subunits. Water soluble polymers may be bonded at specific positions, for example at the amino terminus of the lectomedin products, or randomly attached to one or more side chains of the polypeptide. Additional derivatives include lectomedin species immobilized on a solid support, pin microparticle, or chromatographic resin, as well as lectomedin polypeptides modified to include one or more non-protein labels, tags, or chelating agents.
Also comprehended by the present invention are antibodies (e.g., monoclonal and polyclonal antibodies, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies, and CDR-grafted antibodies, including compounds which include CDR sequences which specifically recognize a polypeptide of the invention) and other binding proteins specific for lectomedin products or fragments thereof. Preferred antibodies of the invention are human antibodies which are produced and identified according to methods described in WO93/11236, published Jun. 20, 1993, which is incorporated herein by reference in its entirety. Antibody fragments, including Fab, Fabxe2x80x2, F(abxe2x80x2)2, and Fv, are also provided by the invention. The term xe2x80x9cspecific forxe2x80x9d indicates that the variable regions of the antibodies of the invention recognize and bind lectomedin polypeptides exclusively (i.e., able to distinguish single lectomedin polypeptides from the family of lectomedin polypeptides despite sequence identity, homology, or similarity found in the family of polypeptides), but may also interact with other proteins (for example, S. aureus protein A or other antibodies in ELISA techniques) through interactions with sequences outside the variable region of the antibodies, and in particular, in the constant region of the molecule. Screening assays to determine binding specificity of an antibody of the invention are well known and routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al. (Eds), Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory; Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognize and bind fragments of the lectomedin polypeptides of the invention are also contemplated, provided that the antibodies are first and foremost specific for, as defined above, lectomedin polypeptides. As with antibodies that are specific for full length lectomedin polypeptides, antibodies of the invention that recognize lectomedin fragments are those which can distinguish single and distinct lectomedin polypeptides from the family of lectomedin polypeptides despite inherent sequence identity, homology, or similarity found in the family of proteins. Antibodies of the invention can be produced using any method well known and routinely practiced in the art.
Non-human antibodies may be humanized by any methods known in the art. In one method, the non-human complementarity determining regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further changes can then be introduced into the antibody framework to modulate affinity or immunogenicity.
Antibodies of the invention are useful for, for example, therapeutic purposes (by modulating activity of lectomedin), diagnostic purposes to detect or quantitate lectomedin, as well as purification of lectomedin. Antibodies are particularly useful for detecting and/or quantitating lectomedin expression in cells, tissues, organs and lysates and extracts thereof, as well as fluids, including serum, plasma, cerebrospinal fluind, urine, sputum, peritoneal fluid, pleural fluid, or pulmonary lavage. Kits comprising an antibody of the invention for any of the purposes described herein are also comprehended. In general, a kit of the invention also includes a control antigen for which the antibody is immunospecific.
Specific binding proteins can be identified or developed using isolated or recombinant lectomedin products, lectomedin variants, or cells expressing such products. Binding proteins are useful for purifying lectomedin products and detection or quantification of lectomedin products in fluid and tissue samples using known immunological procedures. Binding proteins are also manifestly useful in modulating (i.e., blocking, inhibiting or stimulating) biological activities of lectomedin, especially those activities involved in signal transduction.
The DNA and amino acid sequence information provided by the present invention also makes possible the systematic analysis of the structure and function of lectomedins. DNA and amino acid sequence information for lectomedin also permits identification of binding partner compounds with which a lectomedin polypeptide or polynucleotide will interact. Agents that modulate (i.e., increase, decrease, or block) lectomedin activity or expression may be identified by incubating a putative modulator with a lectomedin polypeptide or polynucleotide and determining the effect of the putative modulator on lectomedin activity or expression. The selectivity of a compound that modulates the activity of the lectomedin can be evaluated by comparing its binding activity to one particular lectomedin to its activity to other lectomedin polypeptides. Cell based methods, such as di-hybrid assays to identify DNAs encoding binding compounds and split hybrid assays to identify inhibitors of lectomedin polypeptide interaction with a known binding polypeptide, as well as in vitro methods, including assays wherein a lectomedin polypeptide, lectomedin polynucleotide, or a binding partner are immobilized, and solution assays are contemplated by the invention.
Selective modulators may include, for example, antibodies and other proteins or peptides which specifically bind to a lectomedin polypeptide or a lectomedin-encoding nucleic acid, oligonucleotides which specifically bind to a lectomedin polypeptide or a lectomedin gene sequence, and other non-peptide compounds (e.g., isolated or synthetic organic and inorganic molecules) which specifically react with a lectomedin polypeptide or its underlying nucleic acid. Mutant lectomedin polypeptides which affect the enzymatic activity or cellular localization of the wild-type lectomedin polypeptides are also contemplated by the invention. Presently preferred targets for the development of selective modulators include, for example: (1) regions of the lectomedin polypeptide which contact other proteins, (2) regions that localize the lectomedin polypeptide within a cell, (3) regions of the lectomedin polypeptide which bind substrate, (4) allosteric regulatory binding site(s) of the lectomedin polypeptide, (5) site(s) of the lectomedin polypeptide wherein covalent modification regulates biological activity and (6) regions of the lectomedin polypeptide which are involved in multimerization of lectomedin subunits. Still other selective modulators include those that recognize specific lectomedin encoding and regulatory polynucleotide sequences. Modulators of lectomedin activity may be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which lectomedin activity is known or suspected to be involved.
Lectomedin polypeptides of the invention are amenable to numerous cell based high throughput screening (HTS) assays known in the art, including melanophore assay to investigate receptor-ligand interaction, yeast based assay systems, and mammalian cell expression systems. For a review, see Jayawickreme and Kost, Curr. Opin. Biotechnol. 8:629-634 (1997). Automated and miniaturized HTS assays are also comprehended as described, for example, in Houston and Banks, Curr. Opin. Biotechnol. 8:734-740 (1997).
There are a number of different libraries used for the identification of small molecule modulators, including, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random or designed peptides, oligonucleotides or organic molecules.
Chemical libraries consist of structural analogs of known compounds or compounds that are identified as xe2x80x9chitsxe2x80x9d or xe2x80x9cleadsxe2x80x9d via natural product screening. Natural product libraries are collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) variants thereof. For a review, see Science 282:63-68 (1998). Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries. Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries. For a review of combinatorial chemistry and libraries created therefrom, see Myers, Curr. Opin. Biotechnol. 8:701-707 (1997).
Identification of modulators through use of the various libraries described herein permits modification of the candidate xe2x80x9chitxe2x80x9d (or xe2x80x9cleadxe2x80x9d) to optimize the capacity of the xe2x80x9chitxe2x80x9d to modulate activity.
The scientific value of the information contributed through the disclosures of DNA and amino acid sequences of the present invention is manifest. As one series of examples, knowledge of the sequence of a cDNA for lectomedin makes possible through use of Southern hybridization or polymerase chain reaction (PCR) the identification of genomic DNA sequences encoding lectomedin and lectomedin expression control regulatory sequences such as promoters, operators, enhancers, repressors, and the like. DNA/DNA hybridization procedures carried out with DNA sequences of the invention under moderately to highly stringent conditions are likewise expected to allow the isolation of DNAs encoding allelic variants of lectomedin; allelic variants are known in the art to include structurally related proteins sharing one or more of the biochemical and/or immunological properties specific to lectomedin. Similarly, non-human species genes encoding proteins homologous to human lectomedin can also be identified by Southern and/or PCR analysis; species homologs of the invention are particularly useful in animal models for the study of lectomedin-related disorders. As an alternative, complementation studies can be useful for identifying other human lectomedin products as well as non-human proteins, and DNAs encoding the proteins, sharing one or more biological properties of lectomedin.
Polynucleotides of the invention are also useful in hybridization assays to detect the capacity of cells to express lectomedin. Polynucleotides of the invention may also be the basis for diagnostic methods useful for identifying a genetic alteration(s) in a lectomedin locus that underlies a disease state or states.
Also made available by the invention are anti-sense polynucleotides which recognize and hybridize to polynucleotides encoding lectomedin. Full length and fragment anti-sense polynucleotides are provided. The worker of ordinary skill will appreciate that fragment antisense molecules of the invention include (i) those which specifically recognize and hybridize to lectomedin RNA (as determined by sequence comparison of DNA encoding lectomedin to DNA encoding other known molecules) as well as (ii) those which recognize and hybridize to RNA encoding variants of the lectomedin family of proteins. Antisense polynucleotides that hybridize to RNA encoding other members of the lectomedin family of proteins are also identifiable through sequence comparison to identify characteristic, or signature, sequences for the family of molecules. Anti-sense polynucleotides are particularly relevant to regulating expression of lectomedin by those cells expressing lectomedin mRNA.
Antisense nucleic acids (preferably 10 to 20 base pair oligonucleotides) capable of specifically binding to lectomedin expression control sequences or lectomedin RNA are introduced into cells (e.g., by a viral vector or colloidal dispersion system such as a liposome). The antisense nucleic acid binds to the lectomedin target nucleotide sequence in the cell and prevents transcription or translation of the target sequence. Phosphorothioate and methylphosphonate antisense oligonucleotides are specifically contemplated for therapeutic use by the invention. The antisense oligonucleotides may be further modified by poly-L-lysine, transferrin polylysine, or cholesterol moieties at their 5xe2x80x2 end.
The invention further contemplates methods to modulate lectomedin expression through use of ribozymes. For a review, see Gibson and Shillitoe, Mol. Biotech. 7:125-137 (1997). Ribozyme technology can be utilized to inhibit translation of lectomedin mRNA in a sequence specific manner through (i) the hybridization of a complementary RNA to a target mRNA and (ii) cleavage of the hybridized mRNA through nuclease activity inherent to the complementary strand. Ribozymes can identified by empirical methods but more preferably are specifically designed based on accessible sites on the target mRNA (Bramlage, et al., Trends in Biotech 16:434-438 (1998). Delivery of ribozymes to target cells can be accomplished using either exogenous or endogenous delivery techniques well known and routinely practiced in the art. Exogenous delivery methods can include use of targeting liposomes or direct local injection. Endogenous methods include use of viral vectors and non-viral plasmids.
Ribozymes can specifically modulate expression of lectomedin when designed to be complementary to regions unique to a polynucleotide encoding lectomedin. xe2x80x9cSpecifically modulatexe2x80x9d therefore is intended to mean that ribozymes of the invention recognizes only a polynucleotide encoding lectomedin. Similarly, ribozymes can be designed to modulate expression of all or some of the lectomedin family of proteins. Ribozymes of this type are designed to recognize polynucleotide sequences conserved in all or some of the polynucleotides which encode the family of proteins.
The invention further embraces methods to modulate transcription of lectomedin through use of oligonucleotide-directed triplet helix formation. For a review, see Lavrovsky, et al., Biochem. Mol. Med. 62:11-22 (1997). Triplet helix formation is accomplished using sequence specific oligonucleotides which hybridize to double stranded DNA in the major groove as defined in the Watson-Crick model. Hybridization of a sequence specific oligonucleotide can thereafter modulate activity of DNA-binding proteins, including, for example, transcription factors and polymerases. Preferred target sequences for hybridization include promoter and enhancer regions to permit transcriptional regulation of lectomedin expression.
Oligonucleotides which are capable of triplet helix formation are also useful for site-specific covalent modification of target DNA sequences. Oligonucleotides useful for covalent modification are coupled to various DNA damaging agents as described in Lavrovsky, et al. [supra].
Mutations in a lectomedin gene that results in loss of normal function of the lectomedin gene product may underlie lectomedin-related human disease states. The invention comprehends gene therapy to restore lectomedin activity would thus be indicated in treating those disease states (for example, various forms of cancer described herein). Delivery of a functional lectomedin gene to appropriate cells is effected ex vivo, in situ, or in vivo by use of vectors, and more particularly viral vectors (e.g., adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). See, for example, Anderson, Nature, supplement to vol. 392, no. 6679, pp.25 (1998). For additional reviews of gene therapy technology see Friedmann, Science, 244: 1275-1281 (1989); Verma, Scientific American: 68-84 (1990); and Miller, Nature, 357: 455-460 (1992). Alternatively, it is contemplated that in other human disease states, preventing the expression of or inhibiting the activity of lectomedin will be useful in treating the disease states. It is contemplated that antisense therapy or gene therapy could be applied to negatively regulate the expression of lectomedin.
The invention further embraces pharmaceutical compositions comprising a lectomedin polypeptide of the invention, generally in combination with a pharmaceutically acceptable carrier. The pharmaceutical compositions optionally may include pharmaceutically acceptable (i.e., sterile and non-toxic) liquid, semisolid, or solid diluents that serve as pharmaceutical vehicles, excipients, or media. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma.
The pharmaceutical compositions can be packaged in forms convenient for delivery. The compositions can be enclosed within a capsule, sachet, cachet, gelatin, paper, or other container. These delivery forms are preferred when compatible with entry of the immunogenic composition into the recipient organism and, particularly, when the immunogenic composition is being delivered in unit dose form. The dosage units can be packaged, e.g., in tablets, capsules, suppositories or cachets.
The pharmaceutical compositions may be introduced into the subject to be treated by any conventional method including, e.g., by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., aerosolized drug solutions) or subcutaneous injection (including depot administration for long term release); by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; or by surgical implantation, e.g., embedded under the splenic capsule, brain, or in the cornea. The treatment may consist of a single dose or a plurality of doses over a period of time.
When given parenterally, lectomedin product compositions are generally injected in doses ranging from 1 xcexcg/kg to 100 mg/kg per day, preferably at doses ranging from 0.1 mg/kg to 50 mg/kg per day, and more preferably at doses ranging from 1 to 20 mg/kg/day. The lectomedin product composition may be administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of drug product. Those of ordinary skill in the art will readily optimize effective dosages and administration regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington""s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art without undue experimentation, especially in light of the dosage information and assays disclosed herein, as well as the pharmacokinetic data observed in the human clinical trials discussed above. Appropriate dosages may be ascertained through use of established assays for determining blood levels dosages in conjunction with appropriate dose-response data. The final dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the drug""s specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions.
It will be appreciated that the pharmaceutical compositions and treatment methods of the invention may be useful in the fields of human medicine and veterinary medicine. Thus, the subject to be treated may be a mammal, preferably human, or other animals. For veterinary purposes, subjects include, for example, farm animals including cows, sheep, pigs, horses, and goats, companion animals such as dogs and cats; exotic and/or zoo animals; laboratory animals including mice, rats, rabbits, guinea pigs, and hamsters; and poultry such as chickens, turkeys, ducks and geese.
The present invention is illustrated by the following examples. Example 1 describes identification and characterization of cDNA encoding lectomedin polypeptides. Example 2 relates to expression of recombinant lectomedin. Example 3 described characterization of recombinant lectomedin. Ligand affinity chromatography with immobilized lectomedin is described in Example 4. Example 5 describes Northern analysis of lectomedin expression. Example 5 provides an assessment of tissue distribution of lectomedin in mammalian cell types, while Example 6 describes results from in situ hybridization. The chromosome localization of lectomedin is disclosed in Example 7. Example 8 provides production of polyclonal and monoclonal antibodies specific for lectomedin. Example 9 addresses lectomedin expression.